This invention relates to the formation of conjugates, and in particular to the joining of one species to another at an amino or carbohydrate moiety utilizing a disulfide bond as part of the linkage. More particularly, it relates to substituted 2-iminothiolane hydrohalides and the use of these compounds as crosslinking agents.
The linkage of various compounds to amino or carbohydrate moieties is desirable for several reasons. In the formation of conjugates involving immunoglobulins, for example, linkage at specific regions on the immunoglobulin is often desirable for purposes of maintaining the accessibility of antigen-binding sites or of sites on the Fc chains for complement binding. The species desired for linking to amino or carbohydrate moieties often lack the ability to react directly with these moieties. Hence, some provision must be made for connecting the desired species to the compound containing the reactive moieties.
Immunotoxins are immunoglobulins chemically linked to cytotoxic agents. Generally, immunotoxins are synthesized by covalently linking cell-specific antigen-binding agents to cytotoxic agents. For example, an immunotoxin can be an immunoglobulin antibody linked to a plant or bacterial toxin, such as ricin A chain or abrin A chain. These immunotoxins bind to antigens on the target cell surface, and the cytotoxic agent then enters and kills the cell. Conjugation of the antibody and the toxin is generally by means of crosslinking agents that introduce a disulfide bond between the two proteins. Two disulfide coupling agents commonly used are N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson, et al., Biochem. J. 173:723-737, 1978) and 2-iminothiolane (2IT) (King, et al., Biochemistry 17:1499-1506, 1978). However, the disulfide bond formed by either of these reagents has been reported to be unstable in vivo (Thorpe, et al., Cancer Res. 47:5924-5931, 1987).
Breakdown of this disulfide linkage, with the resulting release of free antibody, is a problem since there is then less immunotoxin available to kill the target cells while at the same time the released antibody can compete with the immunotoxin for the target antigens. Thorpe et al. (supra) have synthesized two coupling agents, sodium S-4-succinimidyloxycarbonyl-.alpha.-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)-toluene (SMPT), which appear to have more stable disulfide linkages in vivo. However, SMBT and SMPT, as well as SPDP have low solubility in water, neutralize the positive charge on modified lysine residues, and exhibit less efficient conjugation than 2IT. Moreover, the reaction of SMBT, SMPT or SPDP with amino or carbohydrate groups cannot be monitored during modification, in contrast to the crosslinking agents of the present invention.